Imatinib mesylate (IM) has revolutionized the treatment of patients with gastrointestinal stromal tumors (GISTs); however, clinical resistance to IM has become a reality, despite the initial efficacy observed. Furthermore, very limited options exist for patients (~20%) that are refractory at the start of treatment. Using clinical pre treatment biopsy samples from a prospective neoadjuvant phase II trial (RTOG 0132), I identified a 32-gene signature that includes KRAB-ZNF 91 subfamily members that can predict response to IM. I subsequently demonstrated that many of these genes were not only predictive of IM response but mediated the drug's activity. In order to determine mechanistically how these ZNFs might be modulating response to IM, RNAi approaches were used to knockdown genes within the predictive signature (including 10 ZNFs) in GIST cells and expression profiling was performed using exon 1.0 ST arrays. This led to the finding that periostin, NEDD9 and TGF23, are universally downregulated following siRNA-mediated knockdown. This is intriguing because TGF23 is a known inducer of periostin and NEDD9, both of which have been reported as frequently overexpressed in a variety of human cancers and indicates that these ZNFs uncovered from these clinical studies may be a master controller over these pathways. Interestingly, hypoxia has been shown to upregulate expression of both NEDD9 and periostin (through the PI3K) leading to increased aggressiveness and enhanced survival of tumor cells. Therefore, it is possible that both periostin and NEDD9 are regulated in a similar mechanism by TGF2 and hypoxia, controlled by ZNFs, to modulate IM response in GIST cells. NEDD9 was also shown to be overexpressed and SRC hyper-activated in an IM-resistant GIST cell line, with knockdown of NEDD9 restoring IM sensitivity. I hypothesize that IM resistance in GISTs may be associated with hypoxia leading to the transactivation of TGF23 by ZNFs causing subsequent induction of periostin and/or NEDD9 and activation of the PI3-K or SRC pathways, respectively. The activation of these potential rescue routes occurs independent of KIT/PDGFRA signaling, thereby overcoming inhibiting activities of IM on pro- survival pathways. My preliminary data suggest that these ZNFs are responsible for regulating this pathway and overexpression of these genes is associated with clinical resistance of GIST patients to IM-based therapy. To test my hypothesis, I propose in Aim 1 to determine if expression of TGF23, NEDD9 and/or periostin in primary GIST samples correlate to IM response (both short and long-term) and ZNF expression. In Aim 2 I will investigate how these ZNFs affect TGF23, NEDD9 and periostin expression and ultimately response to IM. Aim 3 will establish whether targeting the hypoxia-induced TGF23-> periostin-> PI-3K/AKT and/or hypoxia- induced TGF23-> NEDD9-> SRC rescue pathway in vivo will abrogate resistance to IM. The central tenet of this proposal is that targeted agents will become potent when used in combinations that simultaneously block multiple oncogenic pathways, preventing the circumvention of parallel pathways that can act as rescue routes for these tumor cells. The proposed research will be accomplished at Fox Chase Cancer Center (FCCC) in Philadelphia, PA. FCCC is a comprehensive cancer center as designated by the National Cancer Institute, nationally recognized for its leadership in medical, radiation and surgical oncology. FCCC leadership is committed to providing the necessary infrastructure and support for me to successfully complete my proposed studies. I will be mentored by Drs. Margaret von Mehren (Professor & Director, Sarcoma Program), Andrew K. Godwin (Professor & Director of Molecular Oncology at the University of Kansas Medical School (KUMC), and Erica Golemis (Professor & Deputy Chief Scientific Officer at FCCC), esteemed faculty members at FCCC and KUMC and internationally recognized experts in the field of translational research and medical oncology. I will have the opportunity to interact and discuss my research in regular meetings of working groups including the Sarcoma Program, Translational Research Forum, Developmental Therapeutics and bi-monthly lab meetings attended by both Drs. von Mehren and Godwin (by videoconference). My primary goal is to obtain an independent academic career in cancer research. To help accomplish this goal, I have assembled an advisory committee consisting of my mentors and other FCCC faculty members (Drs. Maureen Murphy, Director of Postdoctoral Training Program and Edna Cukierman) in order to review my research progress and provide feedback. During the first two years of funding, I will continue to work closely with my mentors to address the research goals proposed in Aims 1 and 2. During this time I will transition into independence seeking out a tenure-track faculty position. In the last three years of funding, I wil continue the studies proposed in Aims 2 and 3 while establishing my independent research program. As part of my future research objectives, I am interested not only in mechanisms of resistance to novel biologics, such as IM, but in establishing models and challenging existing paradigms to rationally develop combination therapies in order to reduce the burden of cancer. I have already made significant scientific accomplishments in my short career and have established myself to be an important member of the GIST research community. I have authored 10 research articles and scholarly reviews. I have received a number of awards and training fellowships and was selected to orally present my work, the basis of this proposal, at both the AACR and ASCO meetings in 2009. PUBLIC HEALTH RELEVANCE: Despite initial efficacy of imatinib mesylate (IM) in most (~80%) gastrointestinal stromal tumor (GIST) patients, many experience primary/secondary drug resistance, limiting treatment options for these patients. This proposal seeks to identify mechanisms associated with resistance to IM and second line therapies, i.e., sunitinib malate, in GISTs and to subsequently evaluate potential targets and corresponding inhibitors for their ability to improve on current GIST treatment paradigms. This work will establish robust strategies to identify useful combinations in conjunction with predictive biomarkers that identify patients most likely to respond to a specific therapy.